1. Field of the Invention
The invention relates to the fields of cancer biology and nucleic acid biochemistry. Specifically, this invention provides a new method for diagnosing cancer and monitoring angiogenic activity through the amplification and quantitation of a particular gene product indicative of angiogenic activity.
2. Description of Related Art
Over forty target anti-angiogenic agents have been introduced into Phase I, II and III clinical trials in cancer and other non-cancer disease. Cytotoxic agents also possess anti-angiogenic activities. The clinical results from a number of lead anti-angiogenic agents have been disappointing despite their remarkable successes in animal models (Mundhenke et al., 2001). Only until recently, a randomized phase III study showed for the first time that adding bevacezumab, an antiVEGF antibody, to 5-FU, leucovorin, irinotecan (IFL) in metastatic colorectal cancer patients improved tumor response rate, time to tumor progression and overall survival as compared with IFL alone (Hurvitz et al., 2003). Therefore, monitoring and validating anti-angiogenic target response with a defined surrogate(s) would be of paramount clinical importance (Mundhenke et al., 2001; Folkman et al. 2001). Many techniques presently in use are impractical, invasive, and uneconomical.
Microvessel density assay (MVD), the most widely used angiogenesis surrogate, is quantified by counting the density of CD34+ endothelial cells distributed within the tumor (Byrne and Bundred, 2000). MVD has many practical and theoretical limitations for clinical use, however, as it requires direct assessment of microvessels within the tumor tissue. Thus, MVD is invasive and would not be suitable for serial measurements. Furthermore, tumor angiogenesis is enormously heterogeneous, as microvessel density is much higher in the periphery than in the center of an established tumor mass. In addition, MVD overlooks the systemic effects of angiogenic cytokines and, more importantly, endothelial progenitors.
Angiogenesis occurs not only through tumor vessel cooption, but also through mobilization and activation of bone marrow derived endothelial progenitor cells (EPCs) to the sites of active angiogenesis, an increasingly recognized key feature of postnatal angiogenesis, and a feature which MVD assays fail to assess (Asahara et al., 1999). Therefore, EPCs are viable angiogenic surrogates and could be quantified with fluorescence-activated cell sorting techniques (FACS) a using monoclonal antibodies.
However, the FACS procedure has many limitations. For instance, because EPCs are found in low concentrations and also give a poor yield during isolation, FACS assays require up to 50–100 mL of blood per assay. This process can be quite burdensome if serial measurements are required. FACS can be highly variable and subject to poor yield and viability of EPCs, as it is believed that EPCs often undergo apoptosis during isolation procedures, further lowering their recovery. In addition, the FACS procedure is cumbersome, and requires an expensive FACS sorter and an experienced technician to run the machine.
Therefore, there is a need for anti-angiogenic surrogate markers that meet the following specifications for clinical use: (1) they should be non-invasive, accessible and reproducible; (2) they should be feasible for serial measurement and economical; and (3) most importantly, they should mirror the underlying tumor angiogenic activities (Byrne and Bundred, 2000).